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Phosphorylation of myosin light chain kinase by p21-activated kinase PAK2

Z M Goeckeler1, R A Masaracchia, Q Zeng

  • 1Departments of Pathology and Anesthesiology, St. Louis University School of Medicine, St. Louis, Missouri 63104-1028, USA.

The Journal of Biological Chemistry
|April 5, 2000
PubMed

Insights

PAK2 directly inhibits myosin light chain kinase (MLCK) activity by phosphorylating it. This action reduces myosin II RLC phosphorylation, thereby limiting cell contraction and tension development.

Area of Science:

  • Cellular biology
  • Molecular mechanisms of contraction

Background:

  • Myosin II regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) drives smooth muscle and non-muscle cell contraction.
  • Post-translational modifications of MLCK can decrease its activity, reducing RLC phosphorylation and myosin II activation.

Purpose of the Study:

  • To investigate the role of PAK2 in regulating isometric tension development and myosin II RLC phosphorylation in endothelial cells.
  • To determine if PAK2 directly modulates MLCK activity.

Main Methods:

  • Utilized saponin-permeabilized endothelial monolayers to study isometric tension.
  • Assessed myosin II RLC phosphorylation and diphosphorylation.
  • Performed in vitro phosphorylation assays using Cdc42-activated and constitutively active PAK2 on MLCK.
  • Identified phosphorylation sites on MLCK using mass spectrometry.

Main Results:

  • PAK2 significantly reduced isometric tension development by 75% and inhibited myosin II RLC diphosphorylation.
  • PAK2 directly phosphorylated MLCK in vitro, with a stoichiometry of 1.71 mol PO(4)/mol MLCK.
  • PAK2-mediated phosphorylation of MLCK inhibited its ability to phosphorylate myosin II RLC.
  • PAK2 phosphorylated MLCK at Serine 439 and 991; calmodulin binding to MLCK prevented Ser-991 phosphorylation.

Conclusions:

  • PAK2 directly phosphorylates and inhibits MLCK activity.
  • This inhibition of MLCK by PAK2 limits myosin II RLC phosphorylation and subsequent isometric tension development.
  • PAK2 acts as a negative regulator of cell contraction by targeting MLCK.

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